1. Field of the Invention
This invention relates generally to the detection of amplified nucleic acid products, and more specifically to a method of directly detecting the presence or absence of amplified nucleic acid product containing incorporated label molecules.
2. Description of the Background and Related Art
Recent advances in molecular biologic techniques have provided several means for enzymatically amplifying nucleic acid sequences. Currently the most commonly used method, PCR (Polymerase Chain Reaction, Cetus Corporation) involves the use of Taq Polymerase, known sequences as primers, and heating cycles which separate the replicating deoxyribonucleic acid (DNA) strands, in exponentially amplifying a gene of interest. Other amplification methods are currently under development. Examples include the ligase chain reaction (LCR, BioTechnica International) which utilizes DNA ligase, and a probe consisting of two halves of a segment of DNA that is complementary to the sequence of the DNA to be amplified, in mediating DNA amplification. Methods for amplifying genes are not limited to DNA only. Using the enzyme QB replicase (Gene-Trak Systems), and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied, the DNA becomes a template for exponential production of complementary RNA. Nucleic Acid Sequence-Based Amplification (NASBA, Cangene) can be performed on RNA or DNA as the nucleic acid sequence to be repeatedly copied.
Gene amplification techniques are powerful tools with potential to be used for quicker, earlier, and more definitive diagnosis of infectious diseases, genetic disease, and cancers. For example, PCR Protocols: A Guide to Methods and Applications ( .COPYRGT. 1990, Academic Press) describes the use of PCR in the detection of human immunodeficiency virus, hepatitis B virus, genital human papillomaviruses, cytomegalovirus, enteroviruses, B-cell lymphoma, and retinoblastoma. However, some of the amplification techniques such as PCR suffer because they lack fidelity and thus extraneous sequences,i.e. DNA other than the desired sequence, may be amplified. Such possibilities hinder the application to acceptance in diagnostics. Thus, conventional methods for the detection of amplified nucleic acid products require post-amplification specificity by utilizing a post-amplification hybridization technique such as agarose gel electrophoresis with subsequent Southern blot analyses. The following is a list of relevant prior art along with a brief description of each:
U.S. Pat. No. 4,683,195, issued Jul. 28,1987, discloses a process for detecting nucleic acid sequences in a sample suspected to contain the nucleic acid sequences by an amplification process using a chain reaction in which primer extension products are produced. Detection of genetic diseases is described by analyzing the amplified product using radioactive or non-radioactive probes via a Southern blotting technique. In another embodiment a small amount of DNA is amplified to a convenient level first, then further cycles are performed wherein the nucleotide derivatives, which are readily detectable, are incorporated directly into the final product and analyzed by a method such as restriction and electrophoretic separation.
Lundeberg et al., BioTechniques (1991) 10:68-75, describe a method of detecting amplified DNA by biotinylated primers and competitive inhibition using a lac operator sequence incorporated into amplified DNA and detection of the amplified competitor DNA using a fusion protein comprising beta-galactosidase and the LacI repressor.
Dahlen et al., Journal of Microbiology (1991) 29:798-804, describe a time-resolved fluorescence-based hybridization assay. Following amplification by PCR, two oligonucleotide probes, one biotinylated and one labeled with europium, are hybridized to the amplified products. The sandwich hybrids are collected in a microtitration well coated with streptavidin, and the bound europium is measured in a time-resolved fluorometer.
By the term "hapten or target molecule" is meant, for the purposes of the specification and claims, molecules useful as molecular labels which are too small to elicit by themselves an immunological response but are capable of binding with antibodies, and also encompasses small molecules which can serve as molecular labels by virtue of their ability to interact with affinity molecules other than antibody.
U.S. Pat. No. 4,683,195, Lundberg et al. and Dahlen et al., describe methods for the detection of amplified DNA which rely on post-amplification specificity utilizing hybridization with one or more labeled oligonucleotide probes. These detection-by-hybridization systems require the need to a add a label, such as hapten or target molecules, to each primer. Further, a limited amount of label, ranging from one to several molecules per probe, is incorporated into the probe thereby limiting the sensitivity that can be achieved in subsequent detection processes. The method of the present invention to detect amplified nucleic acid products relies on the sensitivity and specificity of the amplification process, since the hapten-labeled nucleotide or target molecule-labeled nucleotides are incorporated directly into the amplified product, rather than post-amplification specificity. For example, the sensitivity and specificity of the amplification process depend critically upon the specificity of the primers and the amplification reaction conditions. Once the primers and amplification reactions have been optimized, these detection-by-hybridization systems are unnecessary. A potential problem for detection of amplified nucleic acid products using the method of present invention is that in an amplification process, such as by PCR, false-positives could result, for example, from physical carryover of amplified DNA from sample to sample in multi-sample analyses. However, physical precautions, procedural safeguards, new equipment and modification of the processes have been, and continue to be, implemented to minimize potential for false-positive results. Thus, the art has now advanced to where it may be possible to omit post-amplification specificity. Further, the method of the present invention incorporates hapten-labeled nucleotide or target molecule-labeled nucleotide directly into the amplified product, in addition to the advantages of omitting pretreatment steps to incorporate label into each primer and the subsequent post-hybridization steps required for detection, there is increased sensitivity because significantly more molecules containing label can be incorporated into the amplified nucleic acid product than can be incorporated onto an oligonucleotide.
Each of the above cited references discloses a diagnostic system utilizing post-amplification specificity for detection of amplified DNA products. Use of agarose gel electrophoresis followed by Southern blot analysis, as disclosed in U.S. Pat. No. 4,683,195, is time consuming, as well as being unsuitable for large scale commercial applications. Lundeberg et al. and Dahlen et al. Disclose a diagnostic system similar to the system described in the present invention in that they all utilize a solid-phase approach to detect immobilized amplified nucleic acid products. In addition to post-hybridization steps, the system disclosed by Lundeburg et al. requires synthesis of a competitor strand of DNA identical to the target DNA to be analyzed. The competitor DNA also requires in-vitro mutagenesis to introduce into that fragment the lac operator sequence. The biotin label is used to bind the competitor strand to the solid support via the affinity molecule streptavidin, but detection is accomplished on the basis of the interaction of the lac operator site of the competitive strand of DNA with the laci repressor protein and .beta.-galactosidase. Note that in the system of the present invention the label, hapten or target molecule, serves a dual purpose by binding the amplified nucleic acid products to the immobilized affinity molecules, as well as interacting with conjugate and substrate in the detection process. In contrast, in the system of Lundeburg et al., the target molecule incorporated into the hybridization probe is only used for binding to the immobilized affinity molecule. The system of the present invention involves direct detection of the amplified nucleic acid product rather than detection of a competitor strand as disclosed by Lundeburg et al. In addition, the system described in the present invention has increased sensitivity, when compared to the system described by Lundeburg et al., since multiple molecules of label are incorporated into the amplified product thereby enhancing the amount of binding to the immobilized affinity molecules, and enhancing calorimetric detection because more incorporated label is available for interaction with conjugate and substrate.
The system described by Dahlen et al. also utilizes a solid phase approach. However, they teach the use of two probes, each containing a different label, in the post-hybridization process leading to detection. One probe is labeled with a target molecule that is used to interact with immobilized affinity molecules, while another probe is labeled with europium (Eu.sup.3+) to be detected in a time-resolved fluorometer. As noted above, the system of the present invention omits the post-hybridization process and utilizes only a single species of label which serves to bind the amplified nucleic acid products to the immobilized affinity molecules, and to interact with conjugate and substrate in the detection process.
In another embodiment of U.S. Pat. No. 4,683,195 is disclosed the incorporation of readily detectable nucleotide derivatives into the final amplified DNA product. The method of the present invention differs in that the step of first amplifying a small amount of DNA to a convenient level, as taught by U.S. Pat. No. 4,683,195, is omitted. In the method of the present invention, the hapten-labeled nucleotide or target molecule--labeled nucleotide is added to the initial reaction thereby obviating the need to stop the reaction. Secondly, the method of the present invention is directed to the detection of amplified nucleic acid products using any one of several available amplification processes and includes the detection of RNA as well as DNA. Only the detection of DNA, amplified by the polymerase chain reaction, is disclosed in U.S. Pat. No. 4,683,195. The method of the present invention provides for an enzyme-linked immunosorbent assay (ELISA)-based system for detection which utilizes a single species of label, hapten or target molecule, that serves to bind the amplified nucleic acid products to immobilized affinity molecules, and to interact with conjugate and substrate in the detection process. Such an assay is well-suited for multi-sample analyses in both clinical and research applications.
Therefore, a method for the detection of amplified nucleic acid products that is direct (not requiring hybridization), simple, quick, and adaptable to multi-sample analyses, is desirable. A method, as such, will greatly facilitate biomedicine in the early detection and diagnoses of infectious agents, cancer, and genetic disease.